The mouse embryo cell line BALB/3T3 clone A31-1-1 was used for quantitative studies on cytotoxicity, mutagenicity and neoplastic transformation induced by different carcinogens. A ouabain resistance (oua-r) mutational assay was\established\for\this\cell\line\with\N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Optimal expression time for oua-r mutations was found to be independent from dose of carcinogen; a linear dose response relationship was obtained for induction of oua-r mutations by MNNG. Split-dose treatments with various intervals between doses were negative for recovery from sublethal damage with MNNG but positive with X-rays; mutation and transformation frequencies were not significantly different after single or split doses of MNNG regardless of time interval between doses. Although MNNG-induced mutation frequencies were found to vary with phases of the cell cycle (maximum in S phase), transformation frequencies did not; this dissociation suggests different underlying mechanisms. A new phenomenon was observed in studies with MNNG exposures maintained for different periods of time after different initial concentrations. Half-life of MNNG in the cultures was about 65 min. The exposure periods required for maximal induction were: 30-60 min for oua-r mutations, about 120 min for cytotoxicity and 120-240 min for transformation; the ratio of transformation to mutation frequencies was within the same order of magnitude at short exposure times and increased to a more than 20-fold ratio at times of 240 min or longer. This temporal dissociation in exposure time required for maximal induction of mutation and transformation supports the hypothesis that transformation is dependent on factors other than a single gene mutation and offers a useful model for investigating the molecular events occurring during this differential time of exposure.